This invention relates to 1-heteroaryl-pyrrolidine, -piperidine and -homopiperidine derivatives and to processes for the preparation of, intermediates used in the preparation of, compositions containing and the uses of, such derivatives.
It has been reported that the immunosuppressant FK-506 promotes neurite outgrowth in vitro in neuronal cell line and culture models (see Lyons et al, Pro. Natl. Acad. Sci., 1994, 91, 3191-95 and Snyder et al, Nature Medicine, 1995, 1, 32-37). WO-A-96/40140, WO-A-96/40633 and WO-A-97/16190 disclose compounds that have neurotrophic activity but which lack inhibitory action at the protein phosphatase calcineurin and therefore which have no immunosuppressive activity. U.S. Pat. No. 5,721,256 discloses sulphonamides, and WO-A-98/13343 and WO-A-98/13355 disclose heterocycles, that have neurotrophic activity but which do not exert any significant immunosuppressive activity. WO-A-92/21313 discloses sulphonamides with immunosuppressive activity.
It has been suggested in WO-A-96/40140 and WO-A-96/40633 that the neurotrophic effect of these compounds is mediated, at least in part, by a high affinity interaction with the FK-506 binding proteins, such as FKBP-12, or FKBP-52. However, the mechanism by which this interaction with FKBP-type immunophilins results in a neurotrophic effect is at present unknown. The range of neurotrophic activity that can be realised through this neurotrophic/non-immunosuppressant class of compounds has been explored and it has been found that axon regeneration can be promoted after facial nerve crush and sciatic nerve crush in the rat. It has also been observed that the functional regeneration of dopamine neurons damaged with the toxin MPTP was promoted by the compounds disclosed therein in mice.
Additionally, it was reported that restoration of striatal innervation in the rat was promoted by the compounds disclosed therein following 6-hydroxydopamine lesioning of dopaminergic neurons (see Hamilton and Steiner, Current Pharmaceutical Design, 1997, 3, 405-428).
It has now been found that the present compounds are neurotrophic agents which have an affinity for FKBP-type immunophilins. In particular, they are potent inhibitors of the enzyme activity and especially of the cis-trans prolyl isomerase (rotamase) activity of FKBP-type immunophilins, particularly the immunophilin FKBP-12. The present compounds do not significantly inhibit the protein phosphatase calcineurin and therefore lack any significant immunosuppressive activity.
The present compounds therefore moderate neuronal degeneration and promote neuronal regeneration and outgrowth and can be used for treating neurological disorders arising from neurodegenerative diseases or other disorders involving nerve damage. The neurological disorders that may be treated include senile dementia (Alzheimer""s disease) and other dementias, amyotrophic lateral sclerosis and other forms of motor neuron disease, Parkinson""s disease, Huntington""s disease, neurological deficits associated with stroke, all forms of degenerative disease affecting the central or peripheral nervous system (e.g. cerebellar-brainstem atrophies, syndromes of progressive ataxias), all forms of muscular dystrophy, progressive muscular atrophies, progressive bulbar muscular atrophy, physical or traumatic damage to the central or peripheral nervous system (e.g. spinal cord), herniated, ruptured or prolapsed intervertebrae disc syndromes, cervical spondylosis, plexus disorders, thoracic outlet syndromes, all forms of peripheral neuropathy (both diabetic and non-diabetic), trigeminal neuralgia, glossopharyngeal neuralgia, Bell""s Palsy, all forms of auto-immune related disease resulting in damage of the central or peripheral nervous system (e.g. multiple sclerosis, myasthenia gravis, Guillain-Barrxc3xa9 syndrome), AIDS related disorders of the nervous system, dapsone ticks, bulbar and retrobulbar affections of the optic nerve (e.g. retinopathies and retrobulbar neuritis), hearing disorders such as tinnitus, and prion diseases.
Preferably, the present compounds can be used for treating senile dementia (Alzheimer""s disease) or another dementia, amyotrophic lateral sclerosis or another form of motor neuron disease, Parkinson""s disease, Huntingdon""s disease, a neurological deficit associated with stroke, physical or traumatic damage to the central or peripheral nervous system (e.g. spinal cord), a peripheral neuropathy (either diabetic or non-diabetic), multiple sclerosis or a hearing disorder such as tinnitus.
The present invention provides a compound of the formula: 
or a pharmaceutically acceptable salt thereof, wherein
A is unbranched C3-C5 alkylene optionally substituted by C1-C6 alkyl;
X is O, S, NH or N(C1-C6 alkyl);
Y is O, S, NH or N(C1-C6 alkyl);
R is a C-linked, 4- to 6-membered ring, non-aromatic, heterocyclic group containing one nitrogen heteroatom, said group being optionally substituted by 1, 2 or 3 substituent(s) each independently selected from C1-C6 alkyl, C2-C6 alkenyl, C3-C7 cycloalkyl, aryl, het, xe2x80x94CO2(C1-C6 alkyl), xe2x80x94CO(het), xe2x80x94CONR5R6 and xe2x80x94CO(aryl), said alkyl and alkenyl being optionally substituted by 1 or 2 substituent(s) each independently selected from C3-C7 cycloalkyl, aryl, het, xe2x80x94O(aryl), xe2x80x94O(C1-C2 alkylene)aryl, xe2x80x94CO(het), xe2x80x94CONR5R6 and xe2x80x94CO(aryl);
R1, R2, R3 and R4 are each independently selected from H, halo, C1-C6 alkyl, C3-C7 cycloalkyl, halo(C1-C6)alkyl, C1-C6 alkoxy, xe2x80x94CONR5R6, C3-C7 cycloalkoxy, C3-C7 cycloalkyl-(C2-C4)alkylene, C3-C7 cycloalkyl(C2-C4)alkoxy and xe2x80x94CO2(C1-C6 alkyl);
R5 and R6 are either each independently selected from H and C1-C6 alkyl or, when taken together, represent unbranched C3-C5 alkylene;
xe2x80x9carylxe2x80x9d means phenyl, optionally substituted by 1, 2 or 3 substituent(s) each independently selected from C1-C6 alkyl, C1-C6 alkoxy, halo, xe2x80x94CONR5R6, halo(C1-C6 alkyl) and xe2x80x94NR5R6; and
xe2x80x9chetxe2x80x9d means a 5- or 6-membered monocyclic, or 8-, 9- or 10-membered bicyclic, ring heterocyclic group containing from 1 to 3 heteroatoms each independently selected from N, O and S, said group being optionally substituted by 1, 2 or 3 substituent(s) each independently selected from C1-C6 alkyl, C1-C6 alkoxy, halo, halo(C1-C6 alkyl), phenyl and xe2x80x94NR5R6.
Throughout the above definitions, xe2x80x9chaloxe2x80x9d means fluoro, chloro, bromo or iodo and alkyl, alkoxy, alkenyl and alkylene groups containing the requisite number of carbon atoms, except where indicated, can be unbranched- or branched-chain.
The pharmaceutically acceptable salts of the compounds of the formula (I) include the acid addition and the base salts thereof.
Suitable acid addition salts are formed from acids which form non-toxic salts and examples are the hydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate, nitrate, phosphate, hydrogen phosphate, acetate, maleate, fumarate, lactate, tartrate, citrate, gluconate, succinate, saccharate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, p-toluenesulphonate and pamoate salts.
Suitable base salts are formed from bases which form non-toxic salts and examples are the sodium, potassium, aluminium, calcium, magnesium, zinc and diethanolamine salts.
For a review on suitable salts see Berge et al, J. Pharm. Sci., 1977, 66, 1-19.
The pharmaceutically acceptable solvates of the compounds of the formula (I) include the hydrates thereof.
Also included within the present scope of the compounds of the formula (I) are polymorphs and radiolabelled derivatives thereof.
A compound of the formula (I) contains one or more asymmetric carbon atoms and therefore exists in two or more stereoisomeric forms. Where a compound of the formula (I) contains an alkenyl or alkenylene group, cis (E) and trans (Z) isomerism may also occur. The present invention includes the individual stereoisomers of the compounds of the formula (I) and, where appropriate, the individual tautomeric forms thereof, together with mixtures thereof.
Separation of diastereoisomers or cis and trans isomers may be achieved by conventional techniques, e.g. by fractional crystallisation, chromatography or H.P.L.C. of a stereoisomeric mixture of a compound of the formula (I) or a suitable salt or derivative thereof. An individual enantiomer of a compound of the formula (I) may also be prepared from a corresponding optically pure intermediate or by resolution, such as by H.P.L.C. of the corresponding racemate using a suitable chiral support or by fractional crystallisation of the diastereoisomeric salts formed by reaction of the corresponding racemate with a suitable optically active acid or base, as appropriate.
Particularly preferred are compounds of the formula: 
wherein R, R1, R2, R3, R4, A, X and Y are as previously defined for a compound of the formula (I).
In the above definitions of a compound of the formula (I) and (IA), the following definitions are preferred.
Preferably, A is 1,4-butylene.
Preferably, X is O, S or NH.
Preferably, X is O or NH.
Preferably, Y is O or NH.
Preferably, Y is NH.
Preferably, R is azetidinyl, pyrrolidinyl or piperidinyl, each optionally substituted as previously defined for R for a compound of the formula (I).
Preferably, R is 3-azetidinyl, 3-pyrrolidinyl, 3-piperidinyl or 4-piperidinyl, each optionally substituted as previously defined for R for a compound of the formula (I).
Preferably, R is azetidinyl, pyrrolidinyl or piperidinyl, each optionally substituted by 1, 2 or 3 substituent(s) each independently selected from C1-C6 alkyl, het, xe2x80x94CO2(C1-C6 alkyl) and xe2x80x94CO(het), said alkyl being optionally substituted by 1 or 2 substituent(s) each independently selected from C3-C7 cycloalkyl, aryl, het, xe2x80x94O(aryl), xe2x80x94O(C1-C2 alkylene)aryl and xe2x80x94CONR5R6.
Preferably, R is 3-azetidinyl, 3-pyrrolidinyl, 3-piperidinyl or 4-piperidinyl, each optionally substituted by 1, 2 or 3 substituent(s) each independently selected from C1-C6 alkyl, het, xe2x80x94CO2(C1-C6 alkyl) and xe2x80x94CO(het), said alkyl being optionally substituted by 1 or 2 substituent(s) each independently selected from C3-C7 cycloalkyl, aryl, het, xe2x80x94O(aryl), xe2x80x94O(C1-C2 alkylene)aryl and xe2x80x94CONR5R6.
Preferably, R is azetidinyl, pyrrolidinyl or piperidinyl, each optionally substituted by 1, 2 or 3 substituent(s) each independently selected from ethyl, 2-pyridyl, tert-butoxycarbonyl, quinolin-2-ylcarbonyl, 2-phenylquinolin-4-ylcarbonyl, 4-methoxyquinolin-2-ylcarbonyl, 6-methoxy-2-phenylquinolin-4-ylcarbonyl, 2-piperidinoquinolin-4-ylcarbonyl, 2-chloroquinolin-4-ylcarbonyl, 1H-benzpyrazol-6-ylcarbonyl, cyclopropylmethyl, phenylmethyl, diphenylmethyl, 2-pyridylmethyl, 3-pyridylmethyl, 4-pyridylmethyl, 2-(2-pyridyl)ethyl, 2-(2-methylimidazol-1-yl)ethyl, (1H-1,2,4-triazol-3-yl)methyl, (2-chloroquinolin-3-yl)methyl, quinolin-4-ylmethyl, quinolin-2-ylmethyl, quinolin-3-ylmethyl, 1-(quinolin-4-yl)ethyl, (2-fluoropyridin-4-yl)methyl, phenoxymethyl, benzyloxymethyl, aminocarbonylmethyl, 2-(aminocarbonyl)ethyl and 3-(aminocarbonyl) phenylmethyl.
Preferably R is 3-azetidinyl, 3-pyrrolidinyl, 3-piperidinyl or 4-piperidinyl, each optionally substituted by 1, 2 or 3 substituent(s) each independently selected from ethyl, 2-pyridyl, tert-butoxycarbonyl, quinolin-2-ylcarbonyl, 2-phenylquinolin-4-ylcarbonyl, 4-methoxyquinolin-2-ylcarbonyl, 6-methoxy-2-phenylquinolin-4-ylcarbonyl, 2-piperidinoquinolin-4-ylcarbonyl, 2-chloroquinolin-4-ylcarbonyl, 1H-benzpyrazol-6-ylcarbonyl, cyclopropylmethyl, phenylmethyl, diphenylmethyl, 2-pyridylmethyl, 3-pyridylmethyl, 4-pyridylmethyl, 2-(2-pyridyl)ethyl, 2-(2-methylimidazol-1-yl)ethyl, (1H-1,2,4-triazol-3-yl)methyl, (2-chloroquinolin-3-yl)methyl, quinolin-4-ylmethyl, quinolin-2-ylmethyl, quinolin-3-ylmethyl, 1-(quinolin-4-yl)ethyl, (2-fluoropyridin-4-yl)methyl, phenoxymethyl, benzyloxymethyl, aminocarbonylmethyl, 2-(aminocarbonyl)ethyl and 3-(aminocarbonyl) phenyl methyl.
Preferably, R1, R2, R3 and R4 are each independently selected from H, halo(C1-C6)alkyl and halo.
Preferably, R1, R2, R3 and R4 are each independently selected from H, fluoro, chloro, bromo and trifluoromethyl.
Preferably, R5and R6 are either each H or, when taken together, are 1,5-pentylene.
Preferably, xe2x80x9carylxe2x80x9d means a phenyl group, optionally substituted by CONR5R6, wherein R5 and R6 are preferably both H.
Preferably, xe2x80x9chetxe2x80x9d means pyridyl, imidazolyl, triazolyl, quinolinyl or benzpyrazolyl, each optionally substituted by 1, 2 or 3 substituent(s) each independently selected from C1-C6 alkyl, C1-C6 alkoxy, halo, phenyl and xe2x80x94NR5R6.
Preferably, xe2x80x9chetxe2x80x9d means pyridyl, imidazolyl, triazolyl, quinolinyl or benzpyrazolyl, each optionally substituted by 1, 2 or 3 substituent(s) each independently selected from methyl, methoxy, fluoro, chloro, phenyl and piperidino.
Preferably, xe2x80x9chetxe2x80x9d means 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazol-1-yl, 1H-1,2,4-triazol-3-yl, quinolin-2-yl, quinolin-3-yl, quinolin-4-yl or 1H-benzpyrazol-6-yl, each optionally substituted by 1, 2 or 3 substituent(s) each independently selected from C1-C6 alkyl, C1-C6 alkoxy; halo, phenyl and xe2x80x94NR5R6.
Preferably, xe2x80x9chetxe2x80x9d means 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazol-1-yl, 1H-1,2,4-triazol-3-yl, quinolin-2-yl, quinolin-3-yl, quinolin-4-yl or 1H-benzpyrazol-6-yl, each optionally substituted by 1,2 or 3 substituent(s) each independently selected from methyl, methoxy, fluoro, chloro, phenyl and piperidino.
Preferably, xe2x80x9chetxe2x80x9d means 2-pyridyl, 3-pyridyl, 4-pyridyl, 1H-1,2,4-triazol-3-yl, quinolin-2-yl, quinolin-3-yl, quinolin-4-yl, 1H-benzpyrazol-6-yl, 2-methylimidazol-1-yl, 2-chloroquinolin-3-yl, 2-phenylquinolin-4-yl, 4-methoxyquinolin-2-yl, 6-methoxy-2-phenylquinolin-4-yl, 2-piperidinoquinolin-4-yl, 2-chloroquinolin-4-yl or 2-fluoropyridin-4-yl.
Preferably, the group of the formula: 
in a compound of the formula (I) is 1,3-benzoxazol-2-yl, 1,3-benzothiazol-2-yl, 1H-benzimidazol-2-yl, 6-bromo-1,3-benzoxazol-2-yl or 6-chloro-1,3-benzothiazol-2-yl.
Particularly preferred examples of the compounds of the formula (I) as described in the Examples section hereafter are:
(2S)-1-(1,3-Benzoxazol-2-yl)-N2-[(3S)-1-benzylpyrrolidin-3-yl]-2-piperidinecarboxamide;
(2S)-1-(1,3-Benzoxazol-2-yl)-N2-[(3S)-1-(2-pyridinylmethyl)pyrrolidin-3-yl]-2-piperidinecarboxamide;
(2S)-1-(1,3-Benzoxazol-2-yl)-N2-[(3S)-1-(3-pyridinylmethyl)pyrrolidin-3-yl]-2-piperidinecarboxamide;
(2S)-1-(1,3-Benzoxazol-2-yl)-N2-[(3S)-1-(4-pyridinylmethyl)pyrrolidin-3-yl]-2-piperidinecarboxamide;
(2S)-N2-{(3S)-1-[3-(Aminocarbonyl)phenylmethyl]pyrroidin-3-yl}-1-(1,3-benzoxazol-2-yl)-2-piperidinecarboxamide;
(2S)-N2-{(3S)-1-[(2-Chlorooquinolin-3-yl)methyl)]pyrrolidin-3-yl}-1-(1,3-benzoxazol-2-yl)-2-piperidinecarboxamide;
(2S)-N2-{(3S)-1-[(Quinolin-3-yl)methyl)]pyrrolidin-3-yl}-1-(1,3-benzoxazol-2-yl)-2-piperidinecarboxamide;
(2S)-N2-{(3S)-1-[(Quinolin-4-yl)methyl)]pyrrolidin-3-yl}-1-(1,3-benzoxazol-2-yl)-2-piperidinecarboxamide;
(2S)-N2-{(3S)-1-[(Quinolin-2-yl)methyl)]pyrrolidin-3-yl}-1-(1,3-benzoxazol-2-yl)-2-piperidinecarboxamide;
(2S)-N2-{(3S)-1-[1-(Quinolin-4-yl)ethyl)]pyrrolidin-3-yl}-1-(1,3-benzoxazol-2-yl)-2-piperidinecarboxamide;
(2S)-N-{(3S)-1-[Quinolin-2-ylcarbonyl]pyrrolidin-3-yl}-1-(1,3-benzoxazol-2-yl)-2-piperidinecarboxamide;
(2S)-N2-{(3S)-1-[2-Phenylquinolin-4-ylcarbonyl]pyrrolidin-3-yl}-1-(1,3-benzoxazol-2-yl)-2-piperidinecarboxamide;
(2S)-N2-{(3S)-1-[4-Methoxyquinolin-2-ylcarbonyl]pyrrolidin-3-yl}-1-(1,3-benzoxazol-2-yl)-2-piperidinecarboxamide;
(2S)-N2-{(3S)-1-[6-Methoxy-2-phenylquinolin-4-ylcarbonyl]pyrrolidin-3-yl}1-(1,3-benzoxazol-2-yl)-2-piperidinecarboxamide;
(2S)-N2-{(3S)-1-[2-Piperidinoquinoin-4-ylcarbonyl]pyrrolidin-3-yl}-1-(1,3-benzoxazol-2-yl)-2-piperidinecarboxamide;
(2S)-N2-{(3S)-1-[2-Chloroquinolin-4-ylcarbonyl]pyrrolidin-3-yl}-1-(1,3-benzoxazol-2-yl)-2-piperidinecarboxamide;
(2S)-N2-{(3S)-1-[1H-benzpyrazol-6-ylcarbonyl]pyrrolidin-3-yl}-1-(1,3-benzoxazol-2-yl)-2-piperidinecarboxamide;
(2S)-N2-[(3S)-1-Benzylpyrrolidin-3-yl]-1-(6-bromo-1,3-benzoxazol-2-yl)-2-piperidinecarboxamide;
(2S)-1-(1,3-Benzoxazol-2-yl)-N2-(1-benzyl-3-piperidinyl)-2-piperidinecarboxamide;
(2S)-1-(1,3-Benzoxazol-2-yl)-N2-(3R,5S)-5-[(benzyloxy)methyl]pyrrolidin-3-yl-2-piperidinecarboxamide hydrochloride; and
1-(1H-1,3-Benzimidazol-2-yl)-N2-[(3S)-1-benzylpyrrolidin-3-yl]-2-piperidinecarboxamide.
The compounds of the formula (I) can be prepared using conventional procedures such as by the following illustrative methods in which R, R1, R2, R3, R4, A, X and Y are as previously defined for a compound of the formula (I) unless otherwise stated.
1) Compounds of the formula (I) wherein X is O or S can be prepared by dehydrative coupling of a compound of the formula: 
wherein X is O or S with a compound of the formula:
xe2x80x83Hxe2x80x94Yxe2x80x94Rxe2x80x83xe2x80x83(III)
Compounds having the formula (II) can be prepared according to the method illustrated in Preparation 3 as detailed herein. Suitable conditions for such preparations use conventional procedures well known to the skilled person such as those referred to in standard texts, e.g. see Advanced Organic Chemistry, Third Edition, Jerry March, 0-56, p. 371-4.
Examples of suitable conditions are as follows:
(a) a compound of the formula (II) may be first converted to an activated ester using 1-hydroxybenzotriazole hydrate and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the presence of a suitable acid acceptor, e.g. triethylamine, and then treated in situ with a compound of the formula (III). The reaction may be carried out in a suitable solvent such as dichloromethane. A catalytic amount of a suitable catalyst, e.g. 4-diethylaminopyridine, may also be used.
(b) compounds of the formula (II) and (III) may be combined with triphenylphosphine and diethyl azodicarboxylate in the presence of a suitable solvent such as tetrahydrofuran.
(c) compounds of the formulae (II) and (III) may be combined with 1,1xe2x80x2-carbonyldiimidazole in the presence of a suitable solvent such as tetrahydrofuran or dichloromethane.
(d) directly heating together compounds of the formulae (II) and (III) optionally in the presence of a suitable solvent, e.g. N,N-dimethylacetamide, cyclopentanol or diphenylether, and optionally in the presence of a suitable acidic catalyst, e.g., where Y is O.
The intermediate compounds of the formula (II) may be prepared by conventional methods, for example, by the route shown in Scheme 1. 
wherein
R7 is C1-C4 alkyl, preferably methyl, or benzyl and
L1 is a suitable leaving group, e.g. halo (preferably, chloro), xe2x80x94SCH3, xe2x80x94SH, xe2x80x94SO2CH3, xe2x80x94SO2CF3, xe2x80x94OSO2CH3 or xe2x80x94OSO2CF3.
The compounds of the formulae (III), (IV) and (VI) may be prepared by conventional procedures.
2) Compounds of the formula (I) where X=NH (i.e. a compound of the formula (IB)) may be prepared by the route shown in Scheme 2, that is by reaction of a compound of the formula (XIIIA) or (XIIIB) with a compound of the formula (III). 
wherein L2 is a suitable leaving group, e.g. as previously defined for L1,
R8 is C1-C4 alkyl (preferably, t-butyl) or benzyl, and
R9 is C1-C4 alkyl or benzyl.
The compounds of the formulae (VIII) and (X) may be prepared by conventional procedures. Reactions using commercially available compounds having the formulae (VIII) and (X) are provided in Preparations 46 and 47.
3) The compounds of the formula (I) wherein X is O or S can be prepared by reaction of a compound of the formula: 
wherein X is O or S, with a compound of the formula:
Hxe2x80x94Yxe2x80x94Rxe2x80x83xe2x80x83(III)
wherein L3 is a suitable leaving group such as
(i) halo, preferably chloro or bromo,
(ii) a group providing an activated ester such as that derived by reaction of a compound of the formula (II) with 1-hydroxybenzotriazole, benzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate, O-(1H-benzotriazol-1-yl)-N,N,Nxe2x80x2,Nxe2x80x2-tetramethyluronium tetrafluoroborate, or pentafluorophenol,
(iii) a group providing a mixed anhydride such as that derived by reaction of a compound of the formula (II) with isobutyl chloroformate,
or (iv) a group providing an imidazolide such as that derived by reaction of a compound of the formula (II) with 1,1xe2x80x2-carbonyldiimidazole.
The reaction may be performed using standard techniques.
Compounds of the formula (XIV) may be prepared by conventional procedures such as from compounds of the formula (II).
4) All compounds of the formula (I) can be prepared by reaction of a compound of the formula: 
with a compound of the formula: 
wherein L3 is as previously defined for L1 for a compound of the formula (VI) and preferably is chloro.
In a preferred procedure, where L3 is chloro, the reaction may be carried out in the presence of a suitable acid acceptor, e.g. ethyidiisopropylamine, and in a suitable solvent, e.g. acetonitrile or N,N-dimethylacetamide, with heating.
Where X=S, the reaction may be conveniently carried out using copper powder, triethylamine hydrochloride and xylene with heating.
The compounds of the formula (XV) may be prepared by conventional procedures similar to those described in Preparation 47 herein. The compounds of the formula (XVI) may be prepared by conventional procedures such as are described in Preparation 47 herein.
It will be appreciated that certain compounds of the formula (I) can be converted to other compounds of the formula (I) by conventional methods, e.g. using standard interconversion techniques. All of the above reactions and the preparations of novel starting materials used in the preceding methods are conventional and appropriate reagents and reaction conditions for their performance or preparation as well as procedures for isolating the desired products will be well-known to those skilled in the art with reference to literature precedents and the Examples and Preparations hereto. It will be appreciated by persons skilled in the art that, within certain of the processes described, the order of the synthetic steps employed may be-varied and will depend inter alia on factors such as the nature of other functional groups present in a particular substrate, the availability of key intermediates and the protecting group strategy (if any) to be employed. Clearly such factors will influence the choice of reagent for use in said synthetic steps.
A pharmaceutically acceptable salt of a compound of the formula (I) may be readily prepared by mixing together solutions of a compound of the formula (I) and the desired acid or base, as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent.
The affinity of the compounds of the formula (I) for FKBP-12 can be determined in vitro in a coupled colorimetric PPlase assay using similar procedures to published methods (e.g. see Kofron, J. L., et al., Biochemistry, 1991, 30, 6127-6134, Zarnt, T., et al., Biochem. J. 1995, 305, 159-164, Holt, D. A., et al., J. Am. Chem. Soc., 1993, 115,9925-9938). In these methods, the cis-trans isomerisation of a hydrophobic amino acid-proline bond in a tetrapeptide substrate (e.g. the phenylalanine-proline bond in N-succinyl-ala-phe-pro-phe-p-nitroanilide [succinyl-AFPF-pNA]) can be determined by monitoring cleavage of pNA from the transPro-containing peptide by an excess of chymotrypsin.
The lC50 (the concentration of the compound of the formula (I) producing 50% inhibition) values were determined using the following assay methodology. Assay buffer (2.175 ml) (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES), 100 mM NaCl, 1 mM dithiothreitol (DTT), pH 8.0) is equilibrated to 10xc2x0 C. in a cuvette. 12.5 xcexcl of a solution of the present compound in DMSO, 250 xcexcl of a 60 mg/ml solution of xcex1-chymotrypsin in 1 mM aqueous hydrochloric acid and then 50 xcexcl of a solution of human recombinant FKBP-12 (4.5 xcexcM) in assay buffer are added and mixed. The reaction is initiated by addition of 12.5 xcexcl of a solution of 20 mM succinyl-AFPF-pNA in DMSO. The absorbance at 390 nM is monitored for one minute collecting data every 0.25 second. Data are fitted with a first order rate equation with offset and the rate constant obtained corrected for the rate of uncatalysed isomerisation of the substrate. The rate constant determined at different inhibitor concentrations (10 nM to 100 xcexcM) is expressed as % inhibition of the control rate constant. The IC50 is estimated using a non-linear least squares curve fitting routine of the sigmoidal dose response data.
Ki,app (the apparent inhibition constant) was determined for the present compounds using the assay procedure described below. Assay buffer (2.175 ml) (50 mM HEPES, 100 mM NaCl, 1 mM DTT, pH 8.0) is equilibrated to 10xc2x0 C. in a cuvette. 12.5 xcexcl of a solution of the present compound in DMSO, 250 xcexcl of a 60 mg/ml solution of xcex1-chymotrypsin in 1 mM aqueous hydrochloric acid and then 50 xcexcL of a solution of human recombinant FKBP-12 (1.5 xcexcM) in assay buffer are added and mixed. The reaction is initiated by adding 12.5 xcexcl of a solution of anhydrous succinyl-ALPF-pNA (100 xcexcM final concentration) in a 400 mM solution of LiCl in trifluoroethanol. The absorbance at 390 nM is monitored for 3 minutes collecting data every 0.5 second. Data are fitted with a first order rate equation with offset and the initial velocity (v) is calculated from the concentration of cis (re leu-pro bond)-succinyl-ALPF-pNA at to and the first order rate constant at different inhibitor concentrations (I). Data in the form vinh/vcontrol v. [I] are fitted with an equation for reversible tight binding inhibition to generate values for Ki,app (see Morrison, J. F., et al, Comments Mol. Cell Biophys., 1985, 2, 347-368). This analysis is used when the Ki,app approaches the concentration of FKBP-12 in the assay (30 nM). Dixon analysis (see Dixon, M., Biochem. J.,1953, 55, 170-171) is used for generating values of Ki,app for less potent compounds. The same methodology is used to generate Ki,app for FKBP52 with the following modifications: Forty microlitres human recombinant FKBP52 (5.2 xcexcM) is substituted for FKBP12 and 2.185 ml assay buffer are used in the assay.
The compounds of the invention have inhibitory activity against the FKBP-12 enzyme. Early experimentation suggests that the compounds of the invention also have inhibitory activity against the FKPB-52 enzyme.
The FKBP-52 enzyme can be expressed and characterised by the methodology described in Peattie, D. A., et al, Proc. Natl. Acad. Sci. USA 1992 November 15; 89 (22):10974-8. The FKPB-52 enzyme is discussed in the following references: Miyata, Y., et al, Proc. Natl. Acad. Sci. USA 1997 December 23; 94(26): 14500-5; Tai, P. K., et al, Biochemistry 1993 August 31; 32(34): 8842-7; Bose, S. et al, Science, 274, 1715-5, 1996 and Czar, M. J., et al, Molecular Endocrinology 9, 1549-1560, 1995.
The neurite outgrowth promoting activity of the compounds of the formula (I) can be determined in explant cultures of embryonic chick dorsal root ganglia. Dorsal root ganglia (DRG) are isolated aseptically according to the method of Bray (see xe2x80x9cCulturing Nerve Cellsxe2x80x9d, Ed. G. Banker and K. Goslin, MIT Press, Cambridge, Mass., 1991, p.119). The individual ganglia were kept in Ca2+/Mg2+-free Tyrodes buffer on ice until a number of ganglia had been collected. Individual ganglia were then transferred into collagen-coated 24-well culture plates containing Neurobasal medium plus B27 supplements and incubated at 37xc2x0 C. in a 5% CO2 atmosphere. The present compound was added after allowing 4 hours for the ganglia to attach. The explants were fixed and stained with Coomassie blue after 24 or 48 hours in culture. For each treatment 4 to 6 ganglia were analysed and scored by estimating the extent of neurite outgrowth relative to the diameter of the explant using image analysis. The present compounds were tested with and without 10 ng/ml nerve growth factor (NGF) present and compared to outgrowth in the presence of 10 ng/ml nerve growth factor alone.
An alternative system for measuring neurite outgrowth promoting activity of FKBP-12 PPlase inhibitors is the SH-SY-5Y neuroblastoma model described by Gold, B. G., et al, in Exp. Neurol., 1997, 147(2), 269-278. Cells are maintained in Dulbecco""s Modified Eagle""s Medium (DMEM) supplemented with 10% Foetal calf serum (FCS), 50 U/ml penicillin, 50 xcexcg/ml streptomycin at 37xc2x0 C. in a 7% CO2 atmosphere. Cells are plated at 1xc3x97106 cells per well and treated for 5 days with 400 nM aphidicolin. Cells are then washed and treated with NGF at 10 ng/mlxc2x1various compound concentrations for 7 days to determine if the compounds promote neurite outgrowth in the presence of suboptimal NGF concentrations (and/or in the absence of NGF). Neurite outgrowth is determined by using image analysis to measure neurite lengths in 20 random fields.
The neurotrophic activity of the present compounds can be evaluated in vivo using the sciatic nerve crush model in rat as a model for peripheral nerve regeneration (see Bridge, P. M., et al. , Experimental Neurology, 1994, 127, 284-290, Medinaceli, L., et al., Expl. Neurology, 1982, 77, 634-643, Gold, B. G.,et al., Restorative Neurology and Neuroscience, 1994, 6, 287-296), the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine models in various species as a model for regeneration in Parkinson""s disease (see Mokry, J., Physiol. Res., 1995, 44(3), 143-150) and fimbria-fornix lesions as a model for regeneration in Alzheimer""s disease (see Cassel, J. C., Duconseille, E., Jeltsch, H. and Will, B., Prog. Neurol., 1997, 51, 663-716).
The compounds of the formula (I) can be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
For example, the compounds of the formula (I) can be administered orally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate or controlled release applications.
Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dicalcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), alginic acid and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc may be included.
Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose or milk sugar as well as high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the compounds of the formula (I) may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
The compounds of the formula (I) can also be injected parenterally, for example, intravenously, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intramuscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.
For oral and parenteral administration to human patients, the daily dosage level of the compounds of the formula (I) will usually be from 1 microgram/kg to 25 mg/kg (in single or divided doses).
Thus tablets or capsules of the compound of the formula (I) may contain from 0.05 mg to 1.0 g of active compound for administration singly or two or more at a time, as appropriate. The physician in any event will determine the actual dosage which will be most suitable for any individual patient and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited and such are within the scope of this invention.
The compounds of formula (I) can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container or a nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A [trade mark] or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA [trade mark]), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the formula (I) and a suitable powder base such as lactose or starch.
Aerosol or dry powder formulations are preferably arranged so that each metered dose or xe2x80x9cpuffxe2x80x9d contains from 20 xcexcg to 20 mg of a compound of the formula (I) for delivery to the patient. The overall daily dose with an aerosol will be in the range of from 20 xcexcg to 20 mg which may be administered in a single dose or, more usually, in divided doses throughout the day.
Alternatively, the compounds of the formula (I) can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder. The compounds of the formula (I) may also be transdermally administered by the use of a skin patch. They may also be administered by the ocular route, particularly for treating neurological disorders of the eye.
For ophthalmic use, the compounds can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.
For application topically to the skin, the compounds of the formula (I) can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
The compounds of the formula (I) can also be administered together with other neurotrophic agents such as neurotrophic growth factor (NGF), glial derived growth factor, brain derived growth factor, ciliary neurotrophic factor and/or neurotrophin-3. The dosage level of the neurotrophic agent will depend upon the neurotrophic effectiveness of the combination and the route of administration used.
It is to be appreciated that all references herein to treatment include curative, palliative and prophylactic treatment.
Thus the invention further provides:
(i) a pharmaceutical composition comprising a compound of the formula (I) or a pharmaceutically acceptable salt or solvate thereof, together with a pharmaceutically acceptable excipient, diluent or carrier;
(ii) a compound of the formula (I), or a pharmaceutically acceptable salt, solvate or composition thereof, for use as a medicament;
(iii) the use of a compound of the formula (I), or of a pharmaceutically acceptable salt, solvate or composition thereof, for the manufacture of a medicament for the treatment of neuronal degeneration;
(iv) the use of a compound of the formula (I), or of a pharmaceutically acceptable salt, solvate or composition thereof, for the manufacture of a medicament for the promotion of neuronal regeneration and outgrowth;
(v) the use of a compound of the formula (I) or of a pharmaceutically acceptable salt, solvate or composition thereof, for the manufacture of a medicament for the treatment of a neurological disease or disorder such as a neurodegenerative disease;
(vi) use as in (v) where the neurological disease or disorder is selected from the group consisting of senile dementia (Alzheimer""s disease) and other dementias, amyotrophic lateral sclerosis and other forms of motor neuron disease, Parkinson""s disease, Huntington""s disease, neurological deficits associated with stroke, all forms of degenerative disease affecting the central or peripheral nervous system (e.g. cerebellar-brainstem atrophies, syndromes of progressive ataxias), all forms of muscular dystrophy, progressive muscular atrophies, progressive bulbar muscular atrophy, physical or traumatic damage to the central or peripheral nervous system (e.g. spinal cord), herniated, ruptured or prolapsed intervertebrae disc syndromes, cervical spondylosis, plexus disorders, thoracic outlet syndromes, all forms of peripheral neuropathy (both diabetic and non-diabetic), trigeminal neuralgia, glossopharyngeal neuralgia, Bell""s Palsy, all forms of auto-immune related disease resulting in damage of the central or peripheral nervous system (e.g. multiple sclerosis, myasthenia gravis, Guillain-Barrxc3xa9 syndrome), AIDS related disorders of the nervous system, dapsone ticks, bulbar and retrobulbar affections of the optic nerve (e.g. retinopathies and retrobulbar neuritis), hearing disorders such as tinnitus, and prion diseases;
(vii) use as (vi) where the neurological disease or disorder is senile dementia (Alzheimer""s disease) or another dementia, amyotrophic lateral sclerosis or another form of motor neuron disease, Parkinson""s disease, Huntington""s disease, a neurological deficit associated with stroke, physical or traumatic damage to the central or peripheral nervous system (e.g. spinal cord), a peripheral neuropathy (either diabetic or non-diabetic), multiple sclerosis or a hearing disorder such as tinnitus;
(viii) a method of treatment of a human to treat neuronal degeneration which comprises treating said human with an effective amount of a compound of the formula (I) or with a pharmaceutically acceptable salt, solvate or composition thereof;
(ix) a method of treatment of a human to promote neuronal regeneration and outgrowth which comprises treating said human with an effective amount of a compound of the formula (I) or with a pharmaceutically acceptable salt, solvate or composition thereof;
(x) a method of treatment of a human to treat a neurological disease or disorder such as a neurodegenerative disease which comprises treating said human with an effective amount of a compound of the formula (I) or with a pharmaceutically acceptable salt, solvate or composition thereof;
(xi) a method as in (x) where the neurological disease or disorder is selected from the group consisting of senile dementia (Alzheimer""s disease) and other dementias, amyotrophic lateral sclerosis and other forms of motor neuron disease, Parkinson""s disease, Huntington""s disease, neurological deficits associated with stroke, all forms of degenerative disease affecting the central or peripheral nervous system (e.g. cerebellar-brainstem atrophies, syndromes of progressive ataxias), all forms of muscular dystrophy, progressive muscular atrophies, progressive bulbar muscular atrophy, physical or traumatic damage to the central or peripheral nervous system (e.g. spinal cord), herniated, ruptured or prolapsed intervertebrae disc syndromes, cervical spondylosis, plexus disorders, thoracic outlet syndromes, all forms of peripheral neuropathy (both diabetic and non-diabetic), trigeminal neuralgia, glossopharyngeal neuralgia, Bell""s Palsy, all forms of auto-immune related disease resulting in damage of the central or peripheral nervous system (e.g. multiple sclerosis, myasthenia gravis, Guillain-Barrxc3xa9 syndrome), AIDS related disorders of the nervous system, dapsone ticks, bulbar and retrobulbar affections of the optic nerve (e.g. retinopathies and retrobulbar neuritis), hearing disorders such as tinnitus, and prion diseases;
(xii) a method as in (xi) where the neurological disease or disorder is senile dementia (Alzheimer""s disease) or another dementia, amyotrophic lateral sclerosis or another form of motor neuron disease, Parkinson""s disease, Huntington""s disease, a neurological deficit associated with stroke, physical or traumatic damage to the central or peripheral nervous system (e.g. spinal cord), a peripheral neuropathy (either diabetic or non-diabetic), multiple sclerosis or a hearing disorder such as tinnitus; and
(xiii) any novel intermediates described herein.
(xiv) the use of a compound of the formula (I) or of a pharmaceutically acceptable salt, solvate or composition thereof, for the manufacture of a medicament for the treatment of a disease resulting from a deficiency or over production of FKBP-12 or FKBP-52.